PROJECT SUMMARY/ABSTRACT Significance: Tubule atrophy underlies progression of acute kidney injury (AKI) to chronic kidney disease. The basis for tubule atrophy after AKI is unknown. We identified a pathology involving RRM2B, alternate regulatory subunit of ribonucleotide reductase (RNR) as a basis for tubule atrophy after AKI. Cell culture data suggest that molecular bypass of RNR by deoxynucleosides can produce deoxynucleoside triphosphates (dNTPs) for DNA synthesis and repair by oxygen independent salvage pathways after AKI to promote tubule recovery. RNR produces dNTPs. Its classical regulatory subunit RRM2 is inhibited by hypoxia, but the hypoxia tolerant RRM2B can substitute for RRM2 to maintain dNTPs during hypoxia to prevent nuclear and mitochondrial DNA damage and preserve cell integrity. RRM2B is induced by hypoxia in cultured cells. Hypoxia mediated increase of mitochondrial superoxide may induce RRM2B as a beneficial adaptation. A regulatory role for mitochondrial superoxide was suggested by our finding that MitoPQ, which increases superoxide selectively in mitochondria, markedly increased cellular RRM2B protein content. The benefits afforded by RRM2B in hypoxic cells would be abrogated if RRM2B is not available. In support, we showed that deletion of RRM2B from cultured tubule cells increases DNA damage during hypoxia, and prevents recovery during reoxygenation. Deoxynucleosides rescued such cells from DNA damage, decreased injury and promoted recovery. We showed also that tubule atrophy after AKI is accompanied by marked RRM2B depletion and DNA damage. Since recovering kidneys are hypoxic, RNR inhibition is expected. In such kidneys RRM2B loss in tubules (rather than an adaptive increase) will have deleterious consequences. Thus, RRM2B loss after AKI may be the cause for tubule atrophy. While the cause for RRM2B decline in tubules after AKi is unclear, its importance for recovery requires investigation. To this end, we have three Specific Aims. Aim 1. We will utilize mouse models of RRM2B deletion and overexpression in tubules to investigate its role in recovery from AKI. Aim 2. We will use cultured tubule cells with RRM2B deletion and overexpression, and deoxynucleoside supplementation, to investigate the role of RNR activity, RRM2B levels and salvage synthesis of dNTPs in cellular responses to hypoxia. To examine the role of mitochondrial oxidants in adaptation to stress, we will use Mito-PQ, a selective inducer of mitochondrial superoxide, to elucidate mechanisms of RRM2B regulation. Aim 3. We will test the efficacy of treatment with deoxynucleosides to promote recovery from ischemic AKI. Impact: The studies we propose will yield new insights into cellular mechanisms that determine successful or failed tubule recovery from AKI and possibly identify deoxynucleosides as novel useful therapeutic agents.